1. Field of the Invention
The invention relates to the field of mass spectrometry, and more particularly to a matrix-assisted laser desorption ionization (MALDI) source for mass spectrometry at about atmospheric pressure. This invention is useful to obtain structural data of compounds especially large complex species.
2. Description of Related Art
A mass spectrometer generally contains the following components:                (1) an optional device to introduce the sample to be analyzed (hereinafter referred to as the “analyte”), such as a liquid or gas chromatograph, direct insertion probe, syringe pump, autosampler or other interfacing device;        (2) an ionization source, which produces ions from the analyte;        (3) at least one analyzer or filter which separates the ions according to their mass-to-charge ratio (m/z);        (4) a detector which measures the abundance of the ions; and        (5) a data processing system that produces a mass spectrum of the analyte.        
There are a number of different ionization sources which are commonly utilized depending upon the type of analyte, including electron impact, chemical ionization, secondary ion mass spectrometry (hereinafter referred to as “SIMS”), fast ion or atom bombardment ionization (hereinafter referred to as “FAB”), field desorption, plasma desorption, laser desorption (hereinafter referred to as “LD”), and matrix-assisted laser desorption ionization (hereinafter referred to as “MALDI”), particle beam, thermospray, electrospray (hereinafter referred to as “ESI”), atmospheric pressure chemical ionization (hereinafter referred to as “APCI”), and inductively coupled plasma ionization.
FAB, ESI and MALDI are particularly useful for the mass analysis and characterization of macromolecules, including polymer molecules, bio-organic molecules (such as peptides, proteins, oligonucleotides, oligosaccharides, DNA, RNA) and small organisms (such as bacteria). MALDI is generally preferred because of its superior sensitivity and greater tolerance of different contaminants such as salts, buffers, detergents and because it does not require a preliminary chromatographic separation.
In the MALDI method, the analyte is mixed in a solvent with small organic molecules having a strong absorption at the laser wavelength (hereinafter referred to as the “matrix”). The solution containing the dissolved analyte and matrix is applied to a metal probe tip or sample stage. As the solvent evaporates, the analyte and matrix co-precipitate out of solution to form a solid solution of the analyte in the matrix on the surface of the probe tip or sample stage. The co-precipitate is then irradiated with a short laser pulse inducing the accumulation of a large amount of energy in the co-precipitate through electronic excitation or molecular vibrations of the matrix molecules. The matrix dissipates the energy by desorption, carrying along the analyte into the gaseous phase. During this desorption process, ions are formed by charge transfer between the photoexcited matrix and the analyte.
The most common type of mass analyzer used with MALDI is the time-of-flight (hereinafter referred to as “TOF”) analyzer. However, other mass analyzers, such as ion trap, ion cyclotron resonance mass spectrometers and quadrupole time-of-flight (QTOF) may be used. These mass analyzers must operate under high vacuum, generally less than 1×10−5 torr. Accordingly, conventional MALDI sources have been operated under high vacuum. This requirement introduces many disadvantages including inter alia:                (1) changing the sample holder requires breaking the vacuum which severely limits sample throughput and generally requires user intervention.        (2) the amount of laser energy used must be kept to a minimum to prevent a broadening of the energy spread of the ions which reduces resolution and capture efficiency;        (3) the positional accuracy and flatness of the sample stage is critical to the mass assignment accuracy and resolution;        (4) it is difficult to test analytes directly on surfaces which are not compatible with high vacuum conditions, including such surfaces as electrophoresis gels and polymer membranes which often shrink under high vacuum conditions; and        (5) tandem mass spectrometry analysis by TOF is relatively difficult and expensive.        
Thus, it would be advantageous to develop a MALDI which operates at about atmospheric pressure yet is still compatible with various mass analyzers to solve the above-described problems. However, no one has heretofore constructed a MALDI source which operates at ambient pressure.
There have been some efforts by others to develop other types of ionization sources which operate at atmospheric pressure.
(a) ESI is a method wherein a solution of the analyte is introduced as a spray into the ion source of the mass spectrometer at atmospheric pressure. The liquid sample emerges from a capillary that is maintained at a few kilovolts relative to its surroundings, whereby the resultant field at the capillary tip charges the surface of the liquid dispersing it by Coulomb forces into a spray of charged droplets. While ESI is a powerful ionization method for macromolecules and small molecules, it is a dynamic method wherein analyte ions are formed in a flowing electrospray. By contrast, MALDI is a pulsed technique wherein ionization of the analyte occurs via a transfer of charge (often a proton) between the absorbing matrix which is irradiated by a pulsed laser of the proper wavelength. Although the MALDI method is inherently more qualitative, its strengths lie in its ability to analyze compounds directly, often in complex biological matrices without extensive sample preparation and/or prior separation. Moreover, MALDI provides ions of low charge states, mostly singly and doubly charged quasimolecular ions, whereas electrospray ionization often produces multiple charge states (charge envelope), particularly for large biomolecules such as proteins.
(b) U.S. Pat. No. 4,527,059 discloses a mass spectrometer having a sample holder mounted on the outside of the vacuum chamber of a mass analyzer. The sample holder exposes the sample to atmospheric pressure or an inert gas environment and is constructed with a polymer carrier film on which the analyte is deposited and which forms part of a wall of the vacuum chamber of the mass spectrometer. The laser is directed onto the analyte causing the analyte to evaporate and simultaneously forming a hole in the carrier film through which the evaporated analyte is transferred into the vacuum chamber. The mass spectrometer uses an ionization source which works on a surface-specific basis, such as SIMS, FAB, and a laser-activated micromass analyzer. This is a laser evaporation/ionization device that is not matrix-assisted.
(c) U.S. Pat. No. 4,740,692 discloses an apparatus using two lasers to produce ions. A first laser is used to vaporize a sample under atmospheric pressure. The second laser is used to ionize the vaporized sample after the vaporized sample enters the vacuum system. While some of the vaporized sample may ionize when the first laser is used under atmospheric pressure, the ions quickly neutralize from interactions with the background gas. This is a laser desorption/ionization device that is not matrix-assisted.
(d) U.S. Pat. No. 5,045,694 discloses a method and instrument for the laser desorption of ions in mass spectrometry. The method teaches the use of matrix compounds which strongly absorb photons from a UV laser beam operating at wavelengths between 200-600 nm, preferably 330-550 nm. Large organic molecules with masses greater than 10,000 Dalton to 200,000 Dalton or higher are analyzed with improved resolution by deflecting low mass (<10,000 Dalton) ions. Both positive and negative ions can be analyzed with reduced fragmentation. The device consists of a TOF mass spectrometer having a MALDI source with a sample probe that is inserted into the vacuum chamber of the mass spectrometer. Analyte ionization occurs by the MALDI process at the sample probe's tip within the vacuum chamber of the mass spectrometer.
(e) U.S. Pat. No. 5,118,937 discloses a process and device for the laser desorption of analyte molecular ions, especially biomolecules. Specific matrices and lasers are employed. The device consists of a TOF mass spectrometer having a MALDI source with a specimen support located within the vacuum chamber of the mass spectrometer or intrinsic to the vacuum chamber wall of the mass spectrometer. Analyte ionization occurs within the vacuum chamber of the mass spectrometer.
(f) U.S. Pat. No. 5,663,561 discloses a device and method for the ionization of analyte molecules at atmospheric pressure by chemical ionization which includes:                (l) codepositing the analyte molecules together with a decomposable matrix material (cellulose trinitrate or trinitrotoluene form a preferred class) on a solid support;        (2) decomposing the matrix with a laser and thereby blasting the analyte molecules into the surrounding gas;        (3) ionizing the analyte molecules within the gas stream by APCI using reactant ions formed in a corona discharge.        
Unlike MALDI, this method requires that the desorption of the analyte be carried out as a separate step from the ionization of the analyte.
Some other U.S. patents of specific interest include but are not limited to:
InventorU.S. Pat. No.Issue DateGray3,944,826Mar. 16, 1976Renner et al.4,209,697Jun. 24, 1980Carr et al.4,239,967Dec. 16, 1980Brunnee et al.4,259,572Mar. 31, 1980Stuke4,686,366Aug. 11, 1987Lee et al.5,070,240Dec. 3, 1991Kotamori et al.5,164,592Nov. 17, 1992Cottrell et al.5,260,571Nov. 9, 1993Buttrill, Jr.5,300,774Apr. 5, 1994Levis et al.5,580,733Dec. 3, 1996Vestal et al.5,625,184Apr. 29, 1997Sakain et al.5,633,496May 27, 1997
Other references of interest include:    M. Karas, et al. International Journal of Mass Spectrometry and Ion Processes, 78, (1987) 53-68. “Matrix-Assisted Ultraviolet Laser Desorption of Non-volatile Compounds”.    K. Tanaka, et al. Rapid Communications in Mass Spectrometry, 2, (1988) 151.    F. Hillenkamp, Analytical Chemistry, 20, (1988), 2299-3000 (Correspondence). “Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10000 Daltons”.    M. Karas, et al. International Journal of Mass Spectrometry and Ion Processes, 92, (1989) 231-242. “UV Laser Matrix Desorption/Ionization Mass Spectrometry of Proteins in the 100000 Dalton Range”.    R. Beavis, et al. “Cinnamic Acid Derivatives as Matrices for Ultraviolet Laser Desorption Mass Spectrometry of Proteins”. Rapid Communications in Mass Spectrometry, 3, (1989) 432-435.    M. Karas, et al. Analytica Chimica Acta, 241, (1990) 175-185. “Principles and applications of matrix-assisted UV-laser desorption/ionization mass spectrometry”.    A. Overberg, et al. Rapid Communications in Mass Spectrometry, 8, (1990) 293-296. “Matrix-assisted Infrared-laser (2.94 μm) Desorption/Ionization Mass Spectrometry of Large Biomolecules”.    B. Spengler, et al., Rapid Communications in Mass Spectrometry, 9, (1990) 301-305. “The Detection of Large Molecules in Matrix-assisted UV-laser Desorption”.    S. Berkenkamp, et al., Proceedings National Academy of Sciences USA, 93, (1996) 7003-7007. “Ice as a matrix for IR-matrix-assisted laser desorption/ionization: Mass spectra from a protein single crystal”.    J. Qin, et al., Analytical Chemistry, 68, (1996) 1784-1791. “A Practical Ion Trap Mass Spectrometer for the Analysis of Peptides by Matrix-Assisted Laser Desorption/Ionization”.    S. Niu, et al., American Society for Mass Spectrometry, 9, (1998) 1-7. “Direct Comparison of Infrared and Ultraviolet Wavelength Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Proteins”.    D. P. Little et al., Analytical Chemistry, 22, (1997), 4540-4546 “MALDI on a Chip: Analysis of Arrays of Low-Femtomole to Subfemtomole Quantities of Synthetic Oligonucleotides and DNA Diagnostic Products Dispensed by a Piezoelectric Pipet.”
Applicants have discovered that a MALDI source may effectively operate at ambient pressure and that such an apparatus is particularly useful for the analysis of organic molecules, such as but not limited to small and large organic compounds, organic polymers, organometallic compounds and the like. Of particular interest are biomolecules and fragments thereof including but not limited to biopolymers such as DNA, RNA, lipids, peptides, protein, carbohydrates—natural and synthetic organisms and fragments thereof such as bacteria, algae, fungi, viral particles, plasmids, cells, and the like.